Methods and apparatuses for purification of gel droplets supporting biological tissue

ABSTRACT

Method and apparatuses for forming gel droplets including biological tissue (e.g., cells), and in particular, methods and apparatuses for removing oil from the gel droplets comprising dissociated cells (including micro-organospheres) are described herein. Although it is beneficial to use oil in the formation of these gel droplets, and particularly micro-organospheres, oil may inhibit growth and survival of the cells within the gel droplets. The methods and apparatuses described herein may permit the removal of oil and may enhance survival and quality of the resulting gel droplets.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional patent applicationNo. 63/070,334, field Aug. 26, 2020, titled “METHODS AND APPARATUSES FORPURIFICIAION OF GEL DROPLETS SUPPORTING BIOLOGICAL TISSUE” and hereinincorporated by reference in its entirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

FIELD

The methods and apparatuses described herein relate to method andapparatuses for forming gel droplets including biological tissue (e.g.,organospheres, e.g., micro-organospheres, organoids, micro-organoids,etc.). Specifically, described herein are methods and apparatuses forforming these gel droplets including removing oil which may otherwiseinhibit or harm the cells within the gel droplets.

BACKGROUND

Model cell and tissue systems are useful for biological and medicalresearch. The most common practice is to derive immortalized cell linesfrom tissue and culture them in two-dimensional (2D) conditions (e.g.,in Petri dish or well plate). However, although immensely useful forbasic research, 2D cell lines do not correlate well with individualpatient response to therapy. In particular, three-dimensional cellculture models are proving particularly helpful in developmentalbiology, disease pathology, regenerative medicine, drug toxicity andefficacy testing, and personalized medicine. For example, spheroids andorganoids are three-dimensional cell aggregates that have been studied.

Multicellular tumor spheroids were first described in the early 70s andobtained by culture of cancer cell lines under non-adherent conditions.Spheroids are typically formed from cancer cell lines as freely floatingcell aggregates in ultra-low attachment plates. Spheroids have beenshown to maintain more stem cell associated properties than 2D cellculture.

Organoids are in-vitro derived cell aggregates that include a populationof stem cells that can differentiate into cell of major cell lineages.Organoids typically have a diameter of more than one mm diameter, andare cultured through passages. It is typically slower to grow and expandorganoid culture than 2D cell culture.

Recently, micro-organoids have been developed, which may be used forrapid and reliable screening, particularly for personalized medicine,such as performing ex-vivo testing of drug response. Micro-organoids maybe smaller, more homogenous is shape and cell number, and may include asmaller number of cells than traditional organoids or tumor spheroids.

However, all of these types of organoids (traditional organoids,spheroids and micro-organospheres), which may be referred to herein as“organospheres,” for convenience, are all typically formed using oil.For example, organoids may be formed by mixing an unpolymerized matrixmaterial (e.g., a substrate basement membrane matrix such as MATRIGEL)with dissociated tissue, such as tumor tissue, and then polymerizingthis mixture into spheres within a stream or bath of immiscible carrierfluid, such as an oil. Although the oil is helpful for forming thespheres, the oil may inhibit the growth of the cells within theorganospheres. The oil may be washed by repeated rinses, however suchrinses are not very effective at removing all of the oil, and may alsorequire longer time periods, during which the cells remain exposed tothe oil.

An emulsification destabilization agent, such as perfluoro octanol (PFO)has been used to remove oil from organospheres. PFO may also disrupt thegrowth and viability of the cells within the organospheres. What isneeded are methods and apparatuses for removal of oil fromorganospheres, including in particular from micro-organospheres. Themethods and apparatuses described herein may address this need.

SUMMARY OF THE DISCLOSURE

Described herein method and apparatuses of forming gel dropletscontaining biological tissue. In particular, described herein aremethods and apparatuses for removing oil from gel droplets shortly orimmediately after they form. The methods and apparatuses describedherein may use a membrane-based demulsification, e.g., using ahydrophobic membrane to remove the oil from the gel droplets. Thesemethods and apparatuses may very rapidly and effectively remove oil(e.g., over 99% of the oil on or around the gel droplets). These methodsand apparatuses may be performed without the use of chemicaldemulsification agents (e.g., perfluoro octanol, PFO), and therefore mayhave substantially lower toxicity to cells within the gel droplets.These methods and apparatuses have been shown to provide gel dropletswith viable biological tissue have an exceptionally high recovery rate(with less than 5% loss of the gel droplets) and may be automated. Theresulting gel droplets may be cultured and/or used immediately or afterculturing for performing one or more assays, including screening, drugtoxicity, etc., assays.

In general, the methods and apparatuses described herein may include theformation of gel droplets including biological tissue. These geldroplets may be referred to herein as organospheres, and may include(but are not limited to) any gel droplet that may be formed with or inoil, such as in particular micro-organospheres, micro-organoids,micro-spheroids, and in some cases organoids and/or spheroids. Any ofthe gel droplets supporting biological tissue (e.g., dissociated cells)described herein may contain cells originating from a patient and/ortissue culture. For example, the cells may be extracted from a smallpatient biopsy, (e.g., for quick diagnostics to guide therapy), fromresected patient tissue, including resected primary tumor or part of adysfunctional organ (e.g., for high-throughput screening), and/or fromalready established PDMCs, including patient-derived xenografts (PDX).These gel droplets may be formed from primary cells that are normal(e.g., normal organ tissue) or from tumor tissue. For example, in somevariations, these methods and apparatuses may form gel droplets fromcancerous tumor biopsy tissue, enabling tailored treatments that canselected using the particular tumor tissue examined.

The gel droplets may be, but are not limited to, micro-organospheres.For example, dissociated primary cells from the patient biopsy may becombined with a fluid matrix material, such as a substrate basementmembrane matrix (e.g., MATRIGEL), to form and gel droplets, such as amicro-organospheres. Micro-organospheres may have a predefined range ofsizes (such as diameters, e.g., between 10 μm and 700 μm and anysub-range therewithin), and initial number of primary cells (e.g.,between 1 and 1000, and in particular lower numbers of cells, such asbetween 1-200). The number of cells and/or the diameter may becontrolled within, e.g., +/−5%, 10%, 15%, 20%, 25%, 30%, etc. Thesemicro-organospheres, when formed as described herein, may be stable foruse and testing within a very short period of time, including withinminutes, hours, or days after being formed, particularly because theymay be free of oil and/or demulsifying agent (e.g., PFO). This may allowfor rapid testing. The gel droplets described herein may more robustlyform 3D cellular structures that replicate and correspond to the tissueenvironment from which they were taken, such as a three-dimensional (3D)tumor microenvironment.

In particular, described herein are methods processing gel dropletsincluding biological tissue (e.g., cells), such as methods of processingorganospheres or micro-organospheres. Any of these methods may include:forming a plurality of gel droplets in an oil, wherein the gel dropletscomprise cells from a dissociated tissue sample distributed within apolymerized sphere of matrix material, the gel droplets having the cellsdistributed therein; and contacting the gel droplets against ahydrophobic membrane so that the oil is removed from the gel dropletsthrough or into the hydrophobic membrane.

The gel droplets may comprise micro-organospheres having a diameter ofbetween 50-500 μm. Any of these methods may include removing at least99% of the oil from the gel droplets when contacting the gel dropletsagainst a hydrophobic membrane.

For example, contacting the gel droplets against a hydrophobic membranemay include passing the gel droplets through a chamber (e.g., tube,channel, etc.) at least partially formed by the hydrophobic membrane.The chamber may comprise a tunnel or tube formed by the hydrophobicmembrane. In any of these methods, negative pressure (e.g., vacuum mayoptionally be applied on one side of the membrane, and/or the solutionincluding the gel droplets may be driven against the membrane.Alternatively, in any of these variations the gel droplets may simply becontacted to the hydrophobic membrane, without requiring the applicationof force, including by applying negative pressure.

For example in some variations, contacting the gel droplets against ahydrophobic membrane comprises eluting the gel droplets into a funnelformed by the hydrophobic membrane. Contacting the gel droplets againsta hydrophobic membrane may comprise filtering the gel droplets againstthe hydrophobic membrane. In general, contacting the gel dropletsagainst a hydrophobic membrane may comprise passing a solution includingthe gel droplets over the hydrophobic membrane.

Any appropriate porous hydrophobic surface, including but not limited toa hydrophobic “membrane” may be used. For example the hydrophobicsurface (e.g., membrane) may have a pore size that is between 0.1 and 5μm (e.g., between about 0.1 and 2 μm, between about 0.2 and 4 μm,between about 0.1 and 1 μm, between about 0.3 and 3 μm, etc.). Thesurface texture of the porous hydrophobic surface may be rough ratherthan smooth.

In general, contacting the gel droplets against a hydrophobic membranemay comprises retaining the gel droplets in an aqueous medium. Forexample, the gel droplets may be retained with an aqueous buffer/mediaon one side of the porous hydrophobic surface while the oil is wicked orremoved into or through the porous hydrophobic surface.

As mentioned above, any of the methods and apparatuses for formingand/or processing gel droplets described herein may be used fororganoids, spheroids or micro-organospheres. For example, in somevariations the gel droplets may each comprises between 1 and 200 of thecells distributed therein.

Any of these methods may include washing the gel droplets on thehydrophobic membrane with an aqueous medium. The gel droplets (e.g., themicroorganospheres) may be washed by adding an aqueous solution (e.g.buffer, such as PBS, culture media, etc.) over the membrane, or addingadditional aqueous solution to the droplets and re-exposing them to thehydrophobic membrane. In some examples, the droplets may be combinedwith an aqueous solution to rinse before contacting the droplets againstthe hydrophobic membrane, then rinsing while on the hydrophobic membranewith additional aqueous solution. Alternatively or additionally, aqueoussolution may be added to the droplets and they may be again exposed tothe hydrophobic membrane (or a new hydrophobic membrane, or new regionof the same hydrophobic membrane). In some example, multiplerinses/washes and contact with one or more hydrophobic membranes may beperformed. In such cases, hydrophobic membranes having differentproperties may be used. The method may include culturing the organizersin a culture medium.

For example, a method of processing gel droplets may include: forming aplurality of gel droplets in an oil, wherein the gel droplets comprisecells from a dissociated tissue sample distributed within a sphere ofmatrix material, the gel droplets having a diameter of between 50 and500 μm with between 1 and 200 of the cells distributed therein; andcontacting the gel droplets against a hydrophobic membrane so that atleast 98% of the oil is removed from the gel droplets on or into thehydrophobic membrane; washing the gel droplets on the hydrophobicmembrane with an aqueous medium; and culturing the organizers in aculture medium.

Any of the method described herein may include combining a dissociatedprimary tissue cells (including, but not limited to cancer/abnormaltissue, normal tissue, etc.) with a liquid matrix material to form anunpolymerized material, and then polymerizing the unpolymerized materialto form gel droplets (e.g., micro-organospheres) within an oil or oilemulsion; these methods may then use a hydrophobic surface or membraneto remove the oil from the gel droplets.

Also described herein are apparatuses configured to perform any of thesemethods. For example, described herein are apparatuses for forming geldroplets, the apparatus comprising: a fluidic processor comprising aplurality of channels, including: a first channel configured to receivea dissociated tissue sample comprising dissociated cells and anunpolymerized matrix material, and a second channel configured toreceive an oil and to intersect with the first channel to formpolymerized gel droplets suspended in the oil; a demulsifying portioncomprising a hydrophobic membrane in fluid communication with thefluidic processor and configured to remove oil from the gel droplets;and an elution channel configured to elute the gel droplets from thedemulsifying portion using an aqueous solution.

When the gel droplets are micro-organospheres, the micro-organospheresmay have diameters that are typically less than about 1000 μm (e.g.,less than about 900 μm, less than about 800 μm, less than about 700 μm,less than about 600 μm, and in particular, less than about 500 μm) indiameter in which the dissociated primary tissue cells are distributed.In any of these gel droplets, the number of dissociated cells may bewithin a predetermined range, as mentioned above (e.g., between about 1and about 500 cells, between about 1-200 cells, between about 1-150cells, between about 100 cells, between about 1-75 cells, between about1-50 cells, between about 1-30 cells, between about 1-20 cells, betweenabout 1-10 cells, between about 5-15 cells, between about 20-30 cells,between about 30-50 cells, between about 40-60 cells, between about50-70 cell, between about 60-80 cells, between about 70-90 cells,between about 80-100 cells, between about 90-110 cells, etc., includingabout 1 cell, about 10 cells, about 20 cells, about 30 cells, about 40cells, about 50 cells, about 60 cells, about 70 cells, etc.). Any ofthese methods may be configured as described herein to produce geldroplets of repeatable size (e.g., having a narrow distribution ofsizes).

The dissociated cells may be freshly biopsied and may be dissociated inany appropriate manner, including mechanical and/or chemicaldissociation (e.g., enzymatic disaggregation by using one or moreenzymes, such as collagenase, trypsin, etc.). The dissociated cells mayoptionally be treated, selected and/or modified. For example, the cellsmay be sorted or selected to identify and/or isolate cells having one ormore characteristics (e.g., size, morphology, etc.). The cells may bemarked (e.g., with one or more markers) that may be used to aid inselection. In some variations the cells may be sorted by a knowncell-sorting technology, including but not limited to microfluidic cellsorting, fluorescent activated cell sorting, magnetic activated cellsorting, etc. Alternatively, the cells may be used without sorting.

In some variations, the dissociated cells may be modified by treatmentwith one or more agents. For example the cells may be geneticallymodified. In some variations the cells may be modified using CRISPR-Cas9or other genetic editing techniques. In some variations the cells may betransfected by any appropriate method (e.g., electroporation, cellsqueezing, nanoparticle injection, magnetofection, chemicaltransfection, viral transfection, etc.), including transfection with ofplasmids, RNA, siRNA, etc. Alternatively, the cells may be used withoutmodification.

One or more additional materials may be combined with the dissociatedcells and fluid (e.g., liquid) matrix material to form the unpolymerizedmixture. For example, the unpolymerized mixture may include additionalcell or tissue types, including support cells. The additional cells ortissue may originate from different biopsy (e.g., primary cells from adifferent dissociated tissue) and/or cultured cells. The additionalcells may be, for example immune cells, stromal cells, endothelialcells, etc. The additional materials may include medium (e.g., growthmedium, freezing medium, etc.), growth factors, support networkmolecules (e.g., collagen, glycoproteins, extracellular matrix, etc.),or the like. In some variations the additional materials may include adrug composition. In some variations the unpolymerized mixture includesonly the dissociated tissue sample (e.g., primary cells) and the fluidmatrix material.

For example, in some variations, these methods may be performed bycombing an unpolymerized matrix mixture with oil (e.g., liquid material)that is immiscible with the unpolymerized material. The method andapparatus may control the size and/or cell density of the gel dropletsby, at least in part, controlling the flow of one or more of theunpolymerized matrix mixture (e.g., the dissociated tissue and fluidmatrix) and the oil with the unpolymerized mixture. Followingpolymerization, typically within the oil, the gel droplets may be washed(e.g., in an aqueous medium, such as a buffered saline (e.g., phosphatebuffered saline) and/or in cell culture medium) to remove some of theoil, and/or the gel droplets may be exposed to a porous hydrophobicsurface, such as in particular a surface of a hydrophobic membrane. Theoil may be removed from the gel droplets through or into the poroushydrophobic surface (e.g., into or through a hydrophobic membrane). Thegel droplets may be separated or removed from the hydrophobic surface byrinsing, washing, flushing, etc. with a buffered solution and/or media.

In some variations, these methods may be performed using a microfluidicsapparatus. In some variations, multiple gel droplets (e.g.,micro-organospheres) may be formed in an oil using one or moremicrofluidic chambers and/or channels, including flow channels. Achamber, channel or other portion may be configured to remove oil fromthe gel droplets. For example, an oil-removing portion may include achannel or chamber including a porous hydrophobic surface (e.g.membrane) onto which the gel droplets may be placed into contact,including by flowing against the hydrophobic surface. The gel dropletsmay be driven (e.g., flowed) against the hydrophobic surface and/orallowed to rest against the hydrophobic surface for some period of time(e.g., x seconds or minutes, such as 1 second, 2 seconds, 3 seconds, 5seconds, 10 seconds, 30 seconds, 60 seconds, 120 seconds, 3 minutes, 4minutes, 5 minutes, etc.). The same apparatus may include multipleparallel channels, including parallel channels for removing oil.

Once the oil has been removed from the gel droplets, the gel dropletsmay be used immediately, and/or may be stored (e.g., frozen) and/or maybe allowed to grow, e.g., by culturing. The gel droplets may be assayedeither before or after culturing and/or may be cryopreserved eitherbefore or after culturing. The gel droplets may be cultured for anyappropriate length of time, but in particular, may be cultured forbetween 1 day and 10 days (e.g., between 1 day and 9 days, between 1 dayand 8 days, between 1 day and 7 days, between 1 day and 6 days, between3 days and 9 days, between 3 days and 8 days, between 3 days and 7 days,etc.). The resulting gel droplets may be essentially free of oil and/orfree of a demulsifying agent, such as PFO.

The matrix material may be a synthetic or non-synthetic unpolymerizedbasement membrane material. In some variations the unpolymerizedbasement material may comprise a polymeric hydrogel. In some variationsthe fluid matrix material may comprise a MATRIGEL. Thus, combining thedissociated tissue sample and the fluid matrix material may comprisecombining the dissociated tissue sample with a basement membrane matrix.The tissue sample may be combined with the fluid matrix material withinsix hours of removing the tissue sample from the patient or sooner(e.g., within about 5 hours, within about 4 hours, within about 3 hours,within about 2 hours, within about 1 hour, etc.).

In any of the methods and apparatuses described herein, rather than, orin addition to, a hydrophobic membrane, a hydrophobic material (beads,surfaces, etc.) may be used to remove the oil. For example, the droplets(e.g., microorganospheres) may be contacted by a surface coated with ahydrophobic material to remove oil, and/or a camber including beadsformed of a hydrophobic material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows one example of an image showing a plurality of gel droplets(in this example, patient-derived micro-organospheres) shortly afterpolymerizing, suspended within a channel containing the oil.

FIG. 2 is an image of a portion of a prototype microfluidics assemblyfor an apparatus for forming gel droplets, and in particularlymicro-organospheres, illustrating the formation of micro-organospheres.

FIG. 3 illustrates a plurality of gel droplets as described herein,shortly after polymerization, suspended in the oil.

FIG. 4A shows on example of a plurality of gel droplets within oilshortly after formation of the gel droplets.

FIG. 4B shows gel droplets from which oil has been removed using anantistatic gun, for comparison to other oil-removing (e.g.,demulsifying) techniques, showing some gel droplets but additional oiland associated debris. Asterisks indicate gel droplets comprisingdissociated cells.

FIG. 4C shows gel droplets following the removal of oil using 10% PFO,as previously described. Asterisks indicate gel droplets comprisingdissociated cells.

FIG. 4D shows gel droplets following removal of the oil using a poroushydrophobic surface (e.g., membrane) as described herein. Asterisksindicate gel droplets comprising dissociated cells.

FIG. 5 schematically illustrates one example of a method for processinggel droplets including removing oil from the gel droplets using a poroushydrophobic surface, as described herein.

FIG. 6 diagrammatically illustrates one example of an apparatusconfigured to form gel droplets, including removal of oil from the geldroplets, as described herein. In FIG. 6 , two separate techniques andstructures for using a porous hydrophobic surface are illustrated (e.g.,a funnel and a tube).

FIG. 7 illustrates one method of manually using a hydrophobic surface toremove oil from gel droplets as described herein.

FIGS. 8A-8D illustrate examples of freshly formed gel droplets (e.g.,micro-organospheres in this example) recovered following the use of ahydrophobic membrane to demulsify the gel droplets. FIG. 8A shows a viewof a plurality of 1 cell/droplet gel droplets at low magnification (4×)objective. FIG. 8B shows a view of a plurality of 20 cell/droplet geldroplets at low magnification (4×) objective. FIG. 8C shows a higher(e.g., 10×) magnification view of gel droplets having 1 cell/dropletfrom which oil was removed using a hydrophobic membrane. FIG. 8D shows ahigher (e.g., 10×) magnification view of gel droplets having 20cells/droplet following removing of the oil as described herein.

FIGS. 9A and 9B show examples of gel droplets having 1 cell/droplet(FIG. 9A) or 20 cells/droplet (FIG. 9B) similar to those shown in FIGS.8A-8B, two days after removal of oil using a hydrophobic membrane asdescribed herein.

FIGS. 10A and 10B show examples of gel droplets having 1 cell/droplet(FIG. 10A) or 20 cells/droplet (FIG. 10B) similar to those shown inFIGS. 8C-8D, two days after removal of oil using a hydrophobic membraneas described herein.

FIGS. 11A and 11B show examples of gel droplets having 1 cell/droplet(FIG. 11A) or 20 cells/droplet (FIG. 11B) similar to those shown inFIGS. 8C-8D, three days after removal of oil using a hydrophobicmembrane as described herein.

FIGS. 12A-12B show examples of gel droplets formed from VERO cellshaving 20 cells/droplet at 4× (FIG. 14A) or 10× (FIG. 14B)magnification, respectively.

FIGS. 13A-13B show examples of gel droplets formed from 293T cellshaving 20 cells/droplet at 4× (FIG. 13A) or 10× (FIG. 13B)magnification, respectively.

FIGS. 14A-14B show examples of gel droplets formed from 293 ACE2 cellshaving 20 cells/droplet at 4× (FIG. 14A) or 10× (FIG. 14B)magnification, respectively.

FIGS. 15A-15B show examples of gel droplets formed from CRC19-106 cellshaving 1 cell/droplet (FIG. 15A) at 4× or 10× (FIG. 15B) magnification,respectively.

FIGS. 16A-16B show examples of gel droplets formed from CRC-1916 cellshaving 20 cells/droplet at 4× (FIG. 16A) or 10× (FIG. 16B)magnification, respectively.

FIGS. 17A-17B show examples of gel droplets formed from CRC19817 cellshaving 1 cell/droplet (FIG. 17A) or 10× (FIG. 17B) magnification,respectively.

FIGS. 18A-18B show examples of gel droplets formed from CRC19187 cellshaving 20 cells/droplet at 4× (FIG. 18A) or 10× (FIG. 18B)magnification, respectively.

FIGS. 19A-19B show examples of gel droplets formed from CRC19245 cellshaving 20 cell/droplet (FIG. 19A) at 4× or 10× (FIG. 19B) magnification,respectively.

FIGS. 20A-20B show examples of gel droplets formed from VERO cellshaving 1 cell/droplet (FIG. 20A) or 20 cells/droplet (FIG. 20B) at 4× or10× magnification, respectively.

FIGS. 21A-21B show examples of gel droplets formed from mouse intestineorganoids having 20 cell/droplet (FIG. 21A) or 20 cells/droplet (FIG.21B) at 4× or 10× magnification, respectively.

FIGS. 22A-22C illustrate examples of micro-organospheres formed asdescribed herein, from induced pluripotent stem cells (iPSCs) using themethods described herein. FIG. 22A shows the droplets(micro-organospheres) shortly after forming, following removal of theoil as described herein. FIG. 22B shows the micro-organospheres threedays after forming. FIG. 22C shows the iPSC micro-organospheres at sevendays.

FIGS. 23A-23F illustrate micro-organospheres (“gel droplets”)successfully formed as described herein from a variety of cadaver(autopsy) tissues. FIG. 23A shows a sample of tissue that may be used asdescribed herein to form the micro-organospheres; in FIG. 23A the tissueis olfactory tissue. FIG. 23B shows an example of a micro-organosphereformed from the tissue shown in FIG. 23A. FIG. 23C shows examples ofmicro-organospheres formed using distal lung tissue. FIG. 23D showsexamples of micro-organospheres formed using tracheal tissue. FIG. 23Eshows examples of micro-organospheres formed using proximal lung tissue.FIG. 23F shows examples of micro-organospheres formed using sinonasalmucosa. FIG. 23H shows examples of micro-organospheres formed usingesophagus tissue. FIG. 23I shows examples of micro-organospheres formedusing intestinal tissue. FIGS. 23J and 23J show examples ofmicro-organospheres formed using liver tissue. The micro-organospheresshown in FIGS. 23B-23K have been cultured for between 7-20 daysfollowing formation and removal of oil, as described herein.

DETAILED DESCRIPTION

In general, described herein are method and apparatuses for making geldroplets, including, for example, the micro-organospheres, thatcomprises a step and/or structure configured to remove oil from the geldroplets by using a porous surface.

The gel droplets described herein may typically be spheres formed fromdissociated primary cells distributed within the base material. Thesegel droplets may be configured as micro-organospheres having a diameterof, e.g., between about 50 μm and about 500 μm (e.g., between about 50μm and about 400 μm, about 50 μm and about 300 μm, about 50 μm and about250 μm, etc.), and may contain between about 1 and 1000 dissociatedprimary cells distributed within the base material (e.g., between about1 and 750, between about 1 and 500, between about 1 and 400, betweenabout 1 and 300, between about 1 and 200, between about 1 and 150,between about 1 and 100, between about 1 and 75, between about 1 and 50,between about 1 and 40, between about 1 and 30, between about 1 and 20,etc.).

The removal of oil using a hydrophobic surface, such as but not limitedto a hydrophobic membrane, may provide gel droplets that may be usedimmediately or cultured for a very brief period of time (e.g., 14 daysor less, 10 days or less, 7 days or less, 5 days or less, etc.) and mayallow the cells within the gel droplets to survive while maintainingmuch, if not all, of the characteristics of the tissue, including tumortissue, from which they were extracted. When using a porous hydrophobicsurface (such as a membrane) to remove oil from the gel droplets, thesurvival rate of the cells within the gel droplets is remarkably high,and the gel droplets may be cultured for days (or weeks) throughmultiple passages, in which the cells will divide, cluster and formstructures similar to the parent tissue.

The gel droplets (e.g., droplet formed Micro-Organospheres) describedherein may be formed, e.g., from patient-derived tumor samples that havebeen dissociated and suspended in a basement matrix (e.g., MATRIGEL).The gel droplets can be patterned onto a microfluidic microwell array,to be incubated, and dosed with drug compounds. This miniaturized assaymaximizes the use of tumor samples and enables more drug compounds to bescreened from a core biopsy at much lower cost per sample.

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

While the terms used herein are believed to be well understood by one ofordinary skill in the art, definitions are set forth herein tofacilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

The term “an unpolymerized mixture” is used herein to refer to acomposition comprising biologically-relevant materials, including adissociated tissue sample and a first fluid matrix material. The fluidmatrix material is typically a material that may be polymerized to forma support or support network for the dissociated tissue (cells)dispersed within it. Once polymerized, the polymerized material may forma hydrogel and may be formed or and/or may include proteins forming thebiocompatible medium, in addition to the cells. A suitable biocompatiblemedium for use in accordance with the presently-disclosed subject mattercan typically be formed from any biocompatible material that is a gel, asemi-solid, or a liquid, such as a low-viscosity liquid, at roomtemperature (e.g., 25° C.) and can be used as a three-dimensionalsubstrate for cells, tissues, proteins, and other biological materialsof interest. Exemplary materials that can be used to form abiocompatible medium in accordance with the presently-disclosed subjectmatter include, but are not limited to, polymers and hydrogelscomprising collagen, fibrin, chitosan, MATRIGEL™ (BD Biosciences, SanJose, Calif.), polyethylene glycol, dextrans including chemicallycrosslinkable or photo-crosslinkable dextrans, and the like, as well aselectrospun biological, synthetic, or biological-synthetic blends. Insome embodiments, the biocompatible medium is comprised of a hydrogel.

The term “hydrogel” is used herein to refer to two- or multi-componentgels comprising a three-dimensional network of polymer chains, wherewater acts as the dispersion medium and fills the space between thepolymer chains. Hydrogels used in accordance with thepresently-disclosed subject matter are generally chosen for a particularapplication based on the intended use of the structure, taking intoaccount the parameters that are to be used to form the gel droplets, aswell as the effect the selected hydrogel will have on the behavior andactivity of the biological materials (e.g., cells) incorporated into thebiological suspensions that are to be placed in the structure. Exemplaryhydrogels of the presently-disclosed subject matter can be comprised ofpolymeric materials including, but not limited to: alginate, collagen(including collagen types I and VI), elastin, keratin, fibronectin,proteoglycans, glycoproteins, polylactide, polyethylene glycol,polycaprolactone, polycolide, polydioxanone, polyacrylates,polyurethanes, polysulfones, peptide sequences, proteins andderivatives, oligopeptides, gelatin, elastin, fibrin, laminin,polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates,polyanhydrides, polyamino acids carbohydrates, polysaccharides andmodified polysaccharides, and derivatives and copolymers thereof as wellas inorganic materials such as glass such as bioactive glass, ceramic,silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, andcombinations of all of the foregoing.

With further regard to the hydrogels used to produce theMicro-Organospheres described herein, in some embodiments, the hydrogelis comprised of a material selected from the group consisting ofagarose, alginate, collagen type I, a polyoxyethylene-polyoxypropyleneblock copolymer (e.g., Pluronic® F127 (BASF Corporation, Mount Olive,N.J.)), silicone, polysaccharide, polyethylene glycol, and polyurethane.In some embodiments, the hydrogel is comprised of alginate.

The gel droplets described herein may also include biologically-relevantmaterials. The phrase “biologically-relevant materials” may describematerials that are capable of being included in a biocompatible mediumas defined herein and subsequently interacting with and/or influencingbiological systems. For example, in some implementations, thebiologically-relevant materials are magnetic beads (i.e., beads that aremagnetic themselves or that contain a material that responds to amagnetic field, such as iron particles) that can be combined as part ofthe unpolymerized material to produce gel droplet that can be used inthe methods and compositions (e.g., for the separation and purificationof gel droplets). As another example, in other implementations, thebiologically-relevant materials may include additional cells, inaddition to the dissociated tissue sample (e.g., biopsy) material. Inthe unpolymerized mixture the dissociated tissue sample and theadditional biologically relevant material in a uniform mixture or as adistributed mixture (e.g., on just one half or other portion of the geldroplet, including just in the core or just in the outer region of theformed gel droplet). In some variations the additionalbiologically-relevant material within the unpolymerized material may besuspended with the dissociated tissue sample in suspension, e.g., priorto polymerization of the droplet forming the gel droplet.

In some variations the biologically relevant material that may beincluded with the dissociated tissue sample (e.g., biopsy) material maycontain a number of cell types, including preadipocytes, mesenchymalstem cells (MSCs), endothelial progenitor cells, T cells, B cells, mastcells, and adipose tissue macrophages, as well as small blood vessels ormicrovascular fragments found within the stromal vascular fraction.

In general, with respect to the dissociated tissue sample, e.g., biopsy,material that is included in the gel droplets described herein, thesetissues may be any appropriate tissue from a patient, typically taken bybiopsy. Although non-biopsy tissue may be used, in general, thesetissues (and the resulting dissociated cells) may be primary cell takenfrom a patient biopsy as described above, e.g., by a needle biopsy.Tissues may be from a healthy tissue biopsy or from cancerous (e.g.,tumor) cell biopsy. The dissociated cells may be incorporated into a geldroplet of the presently-disclosed subject matter, based on the intendeduse of that gel droplet. For example, relevant tissues (e.g.,dissociated biopsy tissue) may typically include cells that are commonlyfound in that tissue or organ (or tumor, etc.). In that regard,exemplary relevant cells that can be incorporated into gel droplets ofthe presently-disclosed subject matter include neurons, cardiomyocytes,myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans,osteocytes, hepatocytes, Kupffer cells, fibroblasts, myoblasts,satellite cells, endothelial cells, adipocytes, preadipocytes, biliaryepithelial cells, and the like. These types of tissues may bedissociated by conventional techniques known in the art. Suitablebiopsied tissue can be derived from: bone marrow, skin, cartilage,tendon, bone, muscle (including cardiac muscle), blood vessels, corneal,neural, brain, gastrointestinal, renal, liver, pancreatic (includingislet cells), lung, pituitary, thyroid, adrenal, lymphatic, salivary,ovarian, testicular, cervical, bladder, endometrial, prostate, vulvaland esophageal tissue. Normal or diseased (e.g., cancerous) tissue maybe used. In some variations, the tissue may arise from tumor tissue,including tumors originating in any of these normal tissues.

Once formed the gel droplets may be cryopreserved and/or cultured.Cultured gel droplets may be maintained in suspension, either static(e.g., in a well, vial, etc.) or in motion (e.g., rolling or agitated).The gel droplet may be cultured using known culturing techniques.Exemplary techniques can be found in, among other places; Freshney,Culture of Animal Cells, A Manual of Basic Techniques, 4th ed., WileyLiss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach,Davis, ed., Oxford University Press, 2002; Animal Cell Culture: APractical Approach, Masters, ed., 2000; and U.S. Pat. Nos. 5,516,681 and5,559,022.

In some variations the gel droplets are formed by forming a droplet ofthe unpolymerized mixture (e.g., in some variations a chilled mixture)of a dissociated tissue sample and a fluid matrix material in an oil.For example, a gel droplet may be formed by combining a stream ofunpolymerized material with one or more streams of the oil to form adroplet. The density of the cells present in the droplet may bedetermined by the dilution of the dissociated material (e.g., cells) inthe unpolymerized material. The size of the gel droplet may correlate tothe size of the droplet formed. In general, the gel droplet is aspherical structure having a stable geometry.

The practice of the presently disclosed subject matter can employ,unless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook,Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press,Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I andII, Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed., 1984;Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984;Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984;Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987;Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), APractical Guide To Molecular Cloning; See Methods In Enzymology(Academic Press, Inc., N. Y.); Gene Transfer Vectors For MammalianCells, J. H. Miller and M. P. Calos, eds., Cold Spring HarborLaboratory, 1987; Methods In Enzymology, Vols. 154 and 155, Wu et al.,eds., Academic Press Inc., N. Y.; Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London, 1987;Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C.Blackwell, eds., 1986.

As used herein a drug composition may include any drug, drug dilution,drug formulation, compositions including multiple drugs (e.g., multipleactive ingredients), drug formulations, drug forms, drug concentrations,combination therapies, and the like. In some variations a drugformulation refers to a formulation comprising a mixture of a drug andone or more inactive ingredients.

During culturing, the cells from the dissociated, biopsied tissue in thegel droplets can aggregate, cluster or assemble within the gel droplets.Aggregates of cells may be highly organized and may form definedmorphology or may be a mass of cells that have clustered or adheredtogether. The organization may reflect the tissue of origin. Although insome variations the gel droplets may contain a single cell type(homotypic), more typically these gel droplets may contain more than onecell type (heterotypic).

As mentioned, the (e.g., biopsy) tissue used to form the gel droplets(e.g., the dissociated tissue) may be derived from a normal or healthybiological tissue, or from a biological tissue afflicted with a diseaseor illness, such as a tissue or fluid derived from a tumor. The tissueused in the gel droplets may include cells of the immune system, such asT lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophagesand dendritic cells. The cells may be stem cells, progenitor cells orsomatic cells. The tissue may be mammalian cells such as human cells orcells from animals such as mice, rats, rabbits, and the like.

Generally, the cells are first dissociated or separated from each otherbefore forming the gel droplets. Dissociation of cells may beaccomplished by any conventional means known in the art. Preferably, thecells are treated mechanically and/or chemically, such as by treatmentwith enzymes. By ‘mechanically’ we include the meaning of disruptingconnections between associated cells, for example, using a scalpel orscissors or by using a machine such as an homogenizer. By‘enzymatically’ we include the meaning of treating the cells with one ormore enzymes disrupt connections between associated cells, including forexample any of collagenase, dispases, DNAse and/or hyaluronidase. One ormore enzymes may be used under different reaction conditions, such asincubation at 37° C. in a water bath or at room temperature.

The dissociated tissue may be treated to remove dead and/or dying cellsand/or cell debris. The removal of such dead and/or dying cells may beaccomplished by any conventional means known to those skilled in theart, for example using beads and/or antibody methods. It is known, forexample, that phosphatidylserine is redistributed from the inner toouter plasma membrane leaflet in apoptotic or dead cells. The use ofAnnexin V-Biotin binding followed by binding of the biotin tostreptavidin magnetic beads enables the separation of apoptotic cellsfrom living cells. Similarly, removal of cell debris may be achieved byany suitable technique in the art, including, for example, filtration.

The dissociated cells may be suspended in a carrier material prior tocombining with the fluid matrix material, and/or the fluid matrixmaterial may be referred to as a carrier material. In some variationsthe carrier material may be a material that has a viscosity level thatdelays sedimentation of cells in a cell suspension prior topolymerization and formation of the gel droplets. A carrier material mayhave sufficient viscosity to allow the dissociated biopsy tissue cellsto remain suspended in the suspension until polymerization. Theviscosity required to achieve this can be optimized by the skilledperson by monitoring the sedimentation rate at various viscosities andselecting a viscosity that gives an appropriate sedimentation rate forthe expected time delay between loading the cell suspension into theapparatus forming the gel droplets forming the gel droplets bypolymerizing the droplets of the unpolymerized material including thecells. In some variations the unpolymerized material may be flowed oragitated by the apparatus even where lower viscosity materials are used,in order to keep the cells in suspension and/or distributed as desired.

As mentioned above, in some variations the unpolymerized mixture,including the dissociated tissue sample and the fluid matrix materialmay include one or more components, e.g., biologically-relevantmaterials. For example, a biologically-relevant material that may beincluded may include any of: an extracellular matrix protein (e.g.fibronectin), a drug (e.g. small molecules), a peptide, or an antibody(e.g., to modulate any of cell survival, proliferation ordifferentiation); and/or an inhibitor of a particular cellular function.Such biologically-relevant materials may be used, for example, toincrease cell viability by reducing cell death and/or activation of cellgrowth/replication or to otherwise mimic the in vivo environment. Thebiologically-relevant materials may include or may mimic one or more ofthe following components: serum, interleukins, chemokines, growthfactors, glucose, physiological salts, amino acids and hormones. Forexample, the biologically-relevant materials may supplement one or moreagents in the fluid matrix material. In some variations, the fluidmatrix material is a synthetic gel (hydrogel) and may be supplemented byone or more biologically-relevant materials. In some variations thefluid matrix is a natural gel. Thus, the gel may be comprised of one ormore extracellular matrix components such as any of collagen,fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin,alginate, agarose and chitosan. For example, MATRIGEL comprisesbioactive polymers that are important for cell viability, proliferation,development and migration. For example, the matrix material may be a gelthat comprises collagen type 1 such as collagen type 1 obtained from rattails. The gel may be a pure collagen type 1 gel or may be one thatcontains collagen type 1 in addition to other components, such as otherextracellular matrix proteins. A synthetic gel may refer to a gel thatdoes not naturally occur in nature. Examples of synthetic gels includegels derived from any of polyethylene glycol (PEG), polyhydroxyethylmethacrylate (PHEMA), polyvinyl alcohol (PVA), poly ethylene oxide(PEO).

Forming Gel Droplets

FIG. 1 illustrates one example of plurality of gel droplets that havebeen formed by combining dissociated tissue cells with a matrix materialwithin an oil carrier to allow the matrix material to polymerize intospherical gel droplets 103, as shown. For example, FIG. 1 illustratesone example of a channel region 139 that is transparent and contains aplurality of gel droplets 103 each containing a predetermined number ofcells 105. The cells may be formed within a microfluidics device, suchas that shown in FIG. 2 , for example. In FIG. 2 , the microfluidicsdevice includes a junction region 237, so that a channel carrying theunpolymerized mixture 211 intersects one or more (e.g., two) channels209 carrying oil that is immiscible with the unpolymerized mixture. Asthe unpolymerized mixture is pressurized to flow at first rate out ofthe first channel 211, the flowing oil in the intersecting channels,209, 209′, permit a predefined amount of the unpolymerized mixture topass before pinching it off to form a droplet 203 that is passed intothe outlet channel 239. Thus, in some variations, a minced (e.g.,dissociated) clinical (e.g., biopsy or resected) sample of tissue, suchas <1 mm in diameter, may be is mixed with a temperature-sensitive gel(i.e. MATRIGEL, at 4 degrees C.) to form the unpolymerized mixture. Thisunpolymerized mixture may be placed into the microfluidic device thatmay generates droplets (e.g., water-in-oil droplets) that are uniform involume and material composition. Simultaneously, the dissociated tumorcells may be partitioned into these droplets. The gel in theunpolymerized material may solidify upon heating (e.g., at 37 degreesC.), and the resulting gel droplets may be formed. These gel droplets(shown here as micro-organospheres) are compatible with traditional 3Dcell culture techniques. FIG. 3 illustrates a plurality of gel droplets305 formed as described above, suspended in the oil 308.

In the exemplary microfluidics chip illustrated above, the junction isshown as a T- or X-junction in which the flow focusing of themicrofluidics forms the controllable size of the gel droplets. In somevariations, rather than a microfluidics chip, the droplets may be formedby robotic micro-pipetting, e.g., into an immiscible fluid and/or onto asolid or gel substrate. Alternatively in some variations the droplets ofunpolymerized material may be formed in the requisite dimensions andreproducibility by micro-capillary generation. Other example oftechniques that may alternatively be used for forming the gel dropletsin the specified size range and reproducibility from the unpolymerizedmaterial may include colloid manipulation, e.g., via external forcessuch as acoustics, magnetics, inertial, electrowetting, orgravitational.

FIG. 4A shows an example of gel droplets in oil formed as describedabove. The gel droplets may be used for an assay immediately, culturedand/or stored (e.g., by cryopreservation). However, it has been foundthat the viability, particularly in culture, is negatively affected byincluding oil with the gel droplets. Further, the presence of oil maymake it difficult or impossible to accurately assay and/or manipulatethe gel droplets. For example, the gel droplets within (or includingsome) oil may clump or cluster together, preventing isolation andmanipulation of individual gel droplets. Thus, it is desirable to removethe oil.

FIGS. 4B and 4C illustrate examples of method that may be used forremoving oil from the gel droplets; these techniques are less thoroughand effective, and may in fact be more complicated, than the methodsdescribed herein using a porous hydrophobic surface. For example, FIG.4B illustrates the gel droplets for which an antistatic gun was used toremove oil. In this example, as can be seen in FIG. 4B, the oil was notcompletely removed, and further, the resulting gel droplets (shown hereby asterisks) are left with oil and debris, possibly resulting fromdestruction of some gel droplets during the demulsification step.Alternatively, FIG. 4C illustrates the use of a chemical demulsificationagent, in this case PFO (10% PFO) used to remove oil from the geldroplets. In this case, the demulsification agent (e.g., PFO) may removethe oil, however, the demulsification agent may also have associatedtoxicity with respect to the gel droplets. Further, the associatedwashing/rinsing steps may add additional time and cost to thepurification and processing steps. In FIG. 4C, the gel droplets arerecovered from the oil phase and resuspended, e.g., into PBS via PFO(perfluoro octanol) and centrifugation. This may separate the immisciblefluid from the gel droplets. Thus, these gel droplets, includingtumor-based organospheres, can be successfully grown

FIG. 4D illustrates gel droplets that have been processed to remove theoil using a porous hydrophobic surface (e.g., membrane) as describedherein. As can be shown in FIG. 4D, the resulting gel droplets (alsoindicated by asterisks) generally appear ‘cleaner,’ with less debris andwith little or no residual oil.

FIG. 5 illustrates one example of a general method of forming/and orprocessing (including removing oil) gel droplets using a poroushydrophobic surface. In FIG. 5 , the method may optionally begin with aprimary tissue sample (or other source of cells to be included in thegel droplets); the tissue sample may be dissociated and/or suspended501. As mentioned above, in some variations the cells may be modified503. The dissociated cells may then be combined with an unpolymerizedmatrix material 505, and streamed into an oil to form the gel dropletswithin the oil; the matrix material with the combined dissociated cells(and any additional components) may then be polymerized, as describedabove, e.g., by increasing the temperature. These steps may generally bepart of a step or multiple steps for forming the gel droplets 507, andall or some of these steps may be automated, e.g., by an apparatus.

As shown in FIG. 5 , the oil may then be removed using a poroushydrophobic surface (e.g., membrane) 502. In some cases it may bepreferable to remove excess oil from the gel droplets first, beforeexposing to the porous hydrophobic surface, as a rough method to removethe bulk of the oil. For example, the gel droplets may be separated fromsome of the oil by spinning, followed by removing of the oil layer(e.g., the bottom layer) by pipetting. Alternatively, the oil and geldroplets suspended therein may be directly placed into contact with theporous hydrophobic surface. In any event, the gel droplets mayde-emulsified using a porous hydrophobic membrane, as described ingreater detail below. This step may remove all or substantially all ofthe oil (e.g., greater than 99%), and may be quick and easy to perform.

Once the oil has been removed, the gel droplets may be separated fromthe porous hydrophobic surface (e.g., membrane), e.g., by washing and/oreluting using a buffer and/or cell culture media 511 into a container.In some cases the gel droplets may be used immediately; alternativelyall or some of the gel droplets may be cultured 513, and/orcryopreserved.

Also described herein are apparatuses that may perform any of thesemethods. For example, FIG. 6 illustrates one example of an apparatusthat may include components to automate all or some of these steps. Forexample, in FIG. 6 , the apparatus may include a microfluidics component601, which may include a plurality of channels (e.g., as part of amicrofluidics chip) and may further include one or more ports forreceiving the unpolymerized matrix, the dissociated cells, and/or theoil, so that they may be combined as shown and described in reference toFIG. 2 , above. Thus, the apparatus may include a fluidic (e.g.,microfluidic) processor comprising a plurality of channels, including afirst channel configured to receive a dissociated tissue samplecomprising dissociated cells and an unpolymerized matrix material, and asecond channel configured to receive an oil and to intersect with thefirst channel to form polymerized gel droplets suspended in the oil. Theprocessor may also include a controller having one or moremicroprocessors, that may control the operation of the apparatus andregulate the formation and processing of the gel droplets. In addition,the apparatus may include a de-emulsifying portion 603, 605 that mayinclude a porous hydrophobic surface (e.g., membrane) 609, 607 in fluidcommunication with the fluidic processor and configured to remove oilfrom the gel droplets. Finally, the apparatus may include an elutionchannel that is configured to elute the gel droplets into one or morecontainers, such as a multi-well plate 615.

In general, the de-emulsifying portion may include any appropriateporous hydrophobic surface, such as a hydrophobic membrane, andparticularly a membrane having pores of between 0.1 μm to 500 μm (e.g.,between 0.1 μm and 400 μm, between 0.1 μm and 300 μm, between 0.1 μm and250 μm, between 0.1 μm and 200 μm, between 0.01 μm and 150 μm, between0.01 μm and 100 μm, etc.). In FIG. 6 , the de-emulsifying portion shows,on the top, a porous hydrophobic membrane 609 formed into a funnel thatmay be used to apply a solution (e.g., an oil-containing solution) ofgel droplets onto/into in order to remove the oil by absorption into orthrough the membrane. FIG. 6 also shown an alternatively embodiment ofthe de-emulsifying portion that includes a channel 605, within which aregion of porous hydrophobic membrane 607 is arranged. The solution ofgel droplets, including oil, may be applied at the first end of thechannel, and the solution, including the gel droplets, may be runthought the channel (e.g. column, etc.) which may be slightly (between 1degree and 30 degrees) at an angle relative to the horizontal, so thatthe fluid may be driven down the channel/column during removal of theoil. In some variations the angle of the hydrophobic surface (formedinto a tunnel 605 in the bottom of FIG. 6 ), may be changed.

For example, FIG. 7 illustrates one example of the removal of oil from aplurality of gel droplets. In FIG. 7 a sheet of hydrophobic membrane,such as a PVDF transfer membrane having a pore size of 0.45 μm, isplaced on a surface (e.g., of a table) and 500 μL of an oil in which aplurality of gel droplets (“Matrigel droplets”) have been formed. Thegel droplets in oil are applied to the surface of the membrane allowingthe oil to absorb into and through the membrane, while the gel dropletsremain on top. In practice, the gel droplets may then be optionally bewashed by applying buffer or media directly onto the gel droplets on themembrane one or more times (e.g., 3×). In some variations the geldroplets may be moved, via the washing, to another portion of themembrane, to allow further removal of the oil by the hydrophobicmembrane. The gel droplets may then be rinsed into a container, shown inthis example as a multi-well plate 715. Analysis of this method hasshown that virtually all (e.g., greater than 99%) of the oil is removedin this manner. Further this type of handling does not negatively affectthe gel droplets.

Indeed, an analysis of fresh gel droplets shortly after processing witha hydrophobic membrane to remove oil from the gel droplets without theuse of a chemical demulsifying agent shows that the overall viability ofthe cells within the processed gel droplets is greater than that of geldroplets processed by other techniques (such as shown in FIG. 4B-4C) toremove oil. In general, the more residual oil left on the gel droplets,the less growth of the cells in the gel droplets was found. Further, themethods described herein were generally significantly faster, requiringless washing and repeating of washing/rinsing steps. Further theresulting gel droplets tended to look clearer, with less geldroplet/cell debris.

For example, FIGS. 8A-8D illustrate one example of gel droplets formedby the method of processing described herein, and allow comparisonbetween low (4×, FIGS. 8A, 8B) and higher (10×, FIGS. 8C and 8D). InFIGS. 8A and 8C, each gel droplets includes one cell and the geldroplets have been cleaned to remove all of the oil, as shown.Similarly, FIGS. 8B and 8D show gel droplets having 20 cells each.

As mentioned above, the gel droplets formed as described herein havebeen seen to have significantly increased viability over time in cultureas compared to de-emulsifying techniques or not de-emulsifying. Thesemethods may also be particularly effective over time in culture, asshown in FIGS. 9A-11B. For example, FIGS. 9A-9B illustrate an example ofa method including the use of a hydrophobic membrane (porous hydrophobicmembrane) as described herein to remove oil from the gel droplets. InFIGS. 9A-9C, gel droplets having 1 (FIG. 9A) or 20 (FIG. 9B) cells (onaverage) are shown at low magnification (e.g., 4×), two days afterforming. As can be seen, the resulting gel droplets are very ‘clean’including the surrounding medium. FIG. 10A-10B show a slightly enlargedview of similar gel droplets after 2 days in culture.

Finally, FIGS. 11A-11B illustrate similar gel droplets after three daysin culture, showing widespread and robust growth within the geldroplets. The gel droplets shown in FIG. 11A-11B are also shown at 10×magnification. As mentioned, the gel droplets have been washed on thehydrophobic membrane in order to remove the oil. This oil may be removedrelatively quickly after forming the Micro-Gel droplets in order toprevent harm to the cells within the Micro-Organosphere.

The methods and apparatuses described herein may generally work withvirtually any cell type for which a gel droplet may formed. For example,FIGS. 12A-12B, 13A-13B, 14A-14B, 15A-15B, 16A-16B, 17A-17B, 18A-18B,19A-19B, 20A-20B and 21A-21B all show different cell or tissue typesused to from gel droplets, for which the methods described herein toremove oil from the gel droplets may be followed. For example, FIGS.12A-12B illustrate VERO cells (20 cells/gel droplet), FIGS. 13A-13Billustrate 293T cells, and FIGS. 14A-14B illustrate gel dropletsincluding 293 ACE2 cells, showing both low magnitude (4×, left) and highmagnitude (10×, right). FIGS. 15A-15B illustrate gel droplets includingCRC19-106 cells at 1 cell/gel droplet, FIGS. 16A-16B also show CRC19-106cells, but at 20 cells/gel droplet, FIGS. 17A-17B show CRC19817 cellshaving 1 cell/gel droplet, and FIGS. 18A-18B illustrate gel dropletsincluding CRC19178 cell having 20 cells/gel droplet, all having oilremoved using hydrophobic membrane, as described herein. FIGS. 19A-19Bshown CRC19245 cells at 1 cell/gel droplet, and FIGS. 20A-20B showCRC19245 cells at 20 cells/gel droplet. Finally, FIGS. 21A-21B show lowand high magnification, respectively, of mouse intestine organoidsincluding 20 cells/gel droplet.

In any of the examples described herein, the method may be a method offorming organospheres (e.g., microorganospheres) from cells that havebeen cultured or isolated (e.g., by dissociation) from tissue. Thesample may be received for processing and may be processed in a verygentle way, including using an automated or semi-automated system. Forexample, the sample may be received and processed in a chilled,temperature-regulated manner, for example, by cooling the temperature ofthe sample (including any media in which the cells are held) and theliquid basement membrane material to a cell processing temperature ortemperature range (e.g. cooled to less than 25, less than 20 degrees C.,less than 19 degrees C., less than 18 degrees C., less than 17 degreesC., less than 16 degrees C., less than 15 degrees C., less than 14degrees C., less than 13 degrees C., less than 12 degrees C., less than11 degrees C., less than 10 degrees C., less than 9 degrees C., lessthan 8 degrees C., less than 7 degrees C., between about 5-25 degreesC., between about 5-20 degrees C., etc.). Thus, the cells may besuspended in an aqueous solution maintained at the cell processingtemperature (or temperature range). The liquid basement membranematerial may also be maintained within the same cell processingtemperature range. The cells may then be combined with a liquid basementmembrane matrix (such as, but not limited to MATRIGEL). The liquidbasement membrane material may be diluted to a predeterminedconcentration by the combination.

The cells in the liquid basement membrane material may then be formedinto droplets by extruding them into an oil. The droplets may be formedby flowing a predetermined amount (and/or at a predetermined flow rate)of the combined cells and liquid basement membrane material into theoil. The oil may be a pool or a stream. Droplets may be formed from thecells in the liquid basement membrane matrix so that each dropletincludes a predetermined amount of cells (e.g., between 1-500 cells,between 1-400 cells, between 1-300 cells, between 1-200 cells, between1-100 cells, etc.). The droplets formed in the oil may then bepolymerized by increasing the temperature. For example the temperaturemay be increased to a polymerization (e.g., to 30 degrees C. or greater,32 degrees C. or greater, 35 degrees C. or greater, between 30-38degrees C., between 32-37 degrees, etc.) to polymerize the gel droplet.The temperature may be increased by increasing the temperature of theoil. In some examples, it may be beneficial to combine the droplet ofcells and liquid basement membrane material in an oil that is at thesame initial temperature and increase the temperature of the surroundingoil after formation of the droplet. In some examples, the droplets ofbasement membrane material including the cells may be added into an oilthat is at a temperature that is higher than the cell processingtemperature (or cell processing temperature range). For example, the oilmay be maintained at the polymerization temperature. Warming thedroplets of the combined cells and the liquid basement membrane matrixmay polymerize the liquid basement membrane matrix material within theoil. Thereafter, the oil may be removed as described herein, and thecells may be cultured to form the organospheres (e.g.,microorganospheres). For example, the droplets may be placed intocontact with a hydrophobic membrane so that the oil is removed from thegel droplets through or into the hydrophobic membrane either beforeadding an aqueous (e.g., culture) media, after rinsing in aqueousculture media, or while adding the aqueous culture media, as describedabove.

This process has proved to be extremely effective at increasing theviability of cells within the resulting organospheres (e.g.,microorganospheres). For example, as compared with other method ofremoving the oil, the viability of even the most sensitive cell typesincreased by greater than 20-50%.

For example, FIGS. 22A-22C illustrate an example of organospheres (e.g.,microorganospheres) formed as described above from induced pluripotentstem cells. Induced pluripotent stem cells (iPSCs) may be extremelyfragile. In FIG. 22A, microorganospheres were formed from iPSCs asdescribed above, in oil, and the oil was removed by contacting themicroorganospheres with a hydrophobic membrane, in this example, a sheetof hydrophobic Polyvinylidene difluoride (PVDF), formed into a surface(e.g., channel, funnel, etc.) through which the microorganospheres maybe flowed, either before or after (or during) the addition of an aqueous(e.g., media) solution. By day 3 (shown in FIG. 22B) the majority of theresulting microorganospheres were viable and the iPSCs within themicroorganospheres had increased in size and number. FIG. 22C shows thesame microorganospheres at day 7. These microorganospheres were able toform “minibrain” structures.

The methods described herein were generally successful for a variety ofdifferent cells including cultured and isolated (e.g., dissociated)cells. For example, FIGS. 23A-23J illustrate the results of the methodsdescribed herein on a variety of different cell types isolated fromhuman autopsy tissue. FIG. 23A shows an example of a sample of tissue(olfactory tissue) removed as part of an autopsy. This tissue wasprocessed as described herein, to dissociate olfactory cells and to formmicroorganospheres from one or more olfactory cell types. For example,FIG. 23B shows one example of a microorganospheres formed from theolfactory cells isolated as described herein. Other cell types similarlyisolated and processed to form microorganospheres as described hereininclude distal lung cells (FIG. 23C), tracheal cells (FIG. 23D),proximal lung cells (FIG. 23E), sinonasal cells (FIG. 23F), esophagealcells (FIG. 23G), intestinal cells (FIG. 23H) and liver cells (FIGS.23I-23J).

Although the methods and apparatuses described herein are described inthe context of gel droplets that include (and support) biologicaltissue, such as dissociated cells, including tumor cells, it should beunderstood that these methods and apparatuses may be used for any geldroplets, with or without biological tissue within the droplet. Inparticular, these methods an apparatuses may be useful for removing oilfrom on or around gel droplets, with or without biological tissue withinthe droplet, including but not limited to gel droplets that are small(e.g., having a diameter of about 2 mm or less (e.g., 1.5 mm or less,1.0 mm or less, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, 0.6 mmor less, 0.5 mm or less, etc., between 50-500 μm, about 50-600 μm, about50-750 μm, about 50-900 μm, about 50 μm to 1 mm, etc.).

Any of the methods (including user interfaces) described herein may beimplemented as software, hardware or firmware, and may be described as anon-transitory computer-readable storage medium storing a set ofinstructions capable of being executed by a processor (e.g., computer,tablet, smartphone, etc.), that when executed by the processor causesthe processor to control perform any of the steps, including but notlimited to: displaying, communicating with the user, analyzing,modifying parameters (including timing, frequency, intensity, etc.),determining, alerting, or the like.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

In general, any of the apparatuses and methods described herein shouldbe understood to be inclusive, but all or a sub-set of the componentsand/or steps may alternatively be exclusive, and may be expressed as“consisting of” or alternatively “consisting essentially of” the variouscomponents, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

What is claimed is:
 1. A method of processing gel droplets containingcells, the method comprising: forming a plurality of gel droplets in anoil, wherein the gel droplets comprise cells from a dissociated tissuesample distributed within a polymerized sphere of matrix material, thegel droplets having the cells distributed therein; and contacting thegel droplets against a hydrophobic membrane so that the oil is removedfrom the gel droplets through or into the hydrophobic membrane.
 2. Themethod of claim 1, wherein the gel droplets comprise micro-organosphereshaving a diameter of between 50-500 μm.
 3. The method of claim 1,wherein contacting the gel droplets against a hydrophobic membranecomprises removing at least 99% of the oil from the gel droplets.
 4. Themethod of claim 1, wherein contacting the gel droplets against ahydrophobic membrane comprises passing the gel droplets through achamber at least partially formed by the hydrophobic membrane.
 5. Themethod of claim 4, wherein the chamber comprises a tunnel or tube formedby the hydrophobic membrane.
 6. The method of claim 1, whereincontacting the gel droplets against a hydrophobic membrane compriseseluting the gel droplets into a funnel formed by the hydrophobicmembrane.
 7. The method of claim 1, wherein contacting the gel dropletsagainst a hydrophobic membrane comprises filtering the gel dropletsagainst the hydrophobic membrane.
 8. The method of claim 1, whereincontacting the gel droplets against a hydrophobic membrane comprisespassing a solution including the gel droplets over the hydrophobicmembrane.
 9. The method of claim 1, wherein the hydrophobic membrane hasa pore size that is between 0.1 and 5 μm.
 10. The method of claim 1,wherein contacting the gel droplets against a hydrophobic membranecomprises retaining the gel droplets in an aqueous medium.
 11. Themethod of claim 1, wherein the gel droplets each comprises between 1 and200 of the cells distributed therein.
 12. The method of claim 1, furthercomprising washing the gel droplets on the hydrophobic membrane with anaqueous medium.
 13. The method of claim 1, further comprising culturingthe gel droplets in a culture medium.
 14. A method of processing geldroplets, the method comprising: forming a plurality of gel droplets inan oil, wherein the gel droplets comprise cells from a dissociatedtissue sample distributed within a sphere of matrix material, the geldroplets having a diameter of between 50 and 500 μm with between 1 and200 of the cells distributed therein; and contacting the gel dropletsagainst a hydrophobic membrane so that at least 98% of the oil isremoved from the gel droplets on or into the hydrophobic membrane;washing the gel droplets on the hydrophobic membrane with an aqueousmedium; and culturing the gel droplets in a culture medium.
 15. Themethod of claim 14, wherein the gel droplets comprisemicro-organospheres having a diameter of between 50-500 μm.
 16. Themethod of claim 14, wherein contacting the gel droplets against ahydrophobic membrane comprises removing at least 99% of the oil from thegel droplets.
 17. The method of claim 14, wherein contacting the geldroplets against a hydrophobic membrane comprises passing the geldroplets through a chamber at least partially formed by the hydrophobicmembrane.
 18. The method of claim 17, wherein the chamber comprises atunnel or tube formed by the hydrophobic membrane.
 19. The method ofclaim 14, wherein contacting the gel droplets against a hydrophobicmembrane comprises eluting the gel droplets into a funnel formed by thehydrophobic membrane.
 20. The method of claim 14, wherein contacting thegel droplets against a hydrophobic membrane comprises filtering the geldroplets against the hydrophobic membrane.
 21. The method of claim 14,wherein contacting the gel droplets against a hydrophobic membranecomprises passing a solution including the gel droplets over thehydrophobic membrane.
 22. The method of claim 14, wherein thehydrophobic membrane has a pore size that is between 0.1 and 5 μm. 23.The method of claim 14, wherein contacting the gel droplets against ahydrophobic membrane comprises retaining the gel droplets in an aqueousmedium.
 24. The method of claim 14, further comprising washing the geldroplets on the hydrophobic membrane with an aqueous medium.